Dipeptides are used in the fields of pharmaceuticals, foods, and various other fields. For example, the dipeptide Asp-Glu has been used for preparation of the diuretic and natriuretic pharmaceutical composition (FR2662359 A1). A pharmaceutical composition, containing dipeptides having agonistic effects on NR1/NR2A and NR1/NR2B subtypes of NMDA receptor is known (JP 2009209131 A). Taste properties of numerous dipeptides have been studied. For example, the dipeptide Asp-Val has sourness taste (Sogame S. and Matsushita I., New Food Ind., 1996, 38(12):44-49 (Japanese)). An excellent saltiness-strengthening agent is known obtained by using a dipeptide containing glutamic acid such as Glu-Ala, Glu-Asp, Glu-Glu, Glu-Ile, Asp-Glu, His-Glu, Trp-Glu, etc. (WO 2009113563 A1).
A variety of methods of producing dipeptides are known, including extraction from protein hydrolysates, chemical synthesis from protected and/or activated amino acids, and enzymatic synthesis using peptidases and protected amino acids (Akabori S. et al., Bull. Chem. Soc. Japan, 1961, 34:739; Monter B. et al., Biotechnol. Appl. Biochem., 1991, 14(2):183-191). A cloning vehicle encoding peptide comprised by the repeating amino acid sequence (Asp-Phe)n has been reported to be useful for production of benzylated and methylated derivatives of dipeptide Asp-Phe (European Patent Application No. 0036258).
The synthesis of dipeptides using chemical and/or chemical-enzymatic approaches requires introduction and removal of protecting groups for functional groups of amino acids to be joined, and isolation of desired product from racemic mixture. The process is thus considered to be disadvantageous from the point of view cost, efficiency, and necessity to discard the concomitant chemicals such as organic solvents, salts, and the like.
Several approaches for enzymatic synthesis of dipeptides and derivatives thereof have been reported, which include a method using reverse reaction of proline iminopeptidase having ability to produce a peptide from an L-amino acid ester and an L-amino acid (Russian Patent No. 2279440), a method using non-ribosomal peptide synthetase (NRPS) (U.S. Pat. Nos. 5,795,738 and 5,652,116; Doekel S. and Marahiel M. A., Chem. Biol., 2000, 7:373-384; Dieckmann R. et al., FEBS Lett., 2001, 498:42-45), a method using aminoacyl-tRNA-synthetase (Japanese Patent Publication Nos.: 58-146539 (1983), 58-209992 (1983), and 59-106298 (1984)), and a method using a mutant protein having the peptide-synthesizing activity (Russian Patent Application 2007127719).
The enzymes belonging to the ATP-dependent carboxylate-amine/thiol α-ligase superfamily have been widely used for production of dipeptides having an α-peptide bond between two L-amino acids. For example, by using the homology search function of SubtiList (http(colon)//genolist(dot)pasteur(dot)fr/SubtiList/), which is a database of the genomic DNA of Bacillus subtilis 168, and the amino acid sequence of D-Ala-D-Ala ligase gene derived from Bacillus subtilis 168, the ywfE gene has been found, which encodes the enzyme capable of synthesizing dipeptides having at the N-terminus the L-amino acid such as, in particular, L-Ala, L-Gly, L-Met, L-Ser, and L-Thr (Tabata K. et al., J. Bacteriol., 2005, 187(15):5195-5202; U.S. Pat. Nos. 7,514,243 and 7,939,302). Despite the YwfE protein (bacilysin synthetase, enzyme classification number (EC) 6.3.2.28) has extremely broad substrate specificity, the enzyme does not accept highly charged amino acids such as L-Lys, L-Arg, L-Glu, and L-Asp, and secondary amines such as L-Pro (Tabata K. et al., J. Bacteriol., 2005, 187(15):5195-5202). Also, a protein encoded by the rhizocticin synthetase gene and having dipeptide-synthesizing activity has been described, which utilizes L-amino acids, Gly, and β-Ala as substrates (U.S. Pat. No. 7,939,294). As confirmed by the liberated phosphoric acid (Pi) as well as TOFMS and NMR analyses, the enzyme places L-Arg and L-Lys on the N-terminus of dipeptide. The Hidden Markov Model (HMM)-based profile analysis revealed five L-amino acids α-ligases (Lals) originating from Treponema denticola ATCC 35405, Photorhabdus luminescence subsp. laumondii TTO1, Streptococcus mutants UA159, Streptococcus pneumoniae TIGR4, and Actinobacillus pleuropneumoniae serovar 1 str. 4074, capable of forming from L-amino acids various peptidyl compounds as proved by the release of phosphoric acid (Senoo A. et al., Biosci. Biotechnol. Biochem., 2010, 74(2):415-418). No dipeptide formation was confirmed in combination of L-Glu or L-Asp with other L-amino acids. A mutant protein having the peptide-synthesizing activity has been confirmed by HPLC using standard samples to form dipeptides bearing L-Met at the N-terminus (Russian Patent Application 2007127719). The in silico screening performed with the help of the NCBI's BLAST service (http(colon)//www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/) and based on the amino acid sequence of Lal from B. subtilis (BsLal) has revealed a protein RSp1486a from Ralstonia solanacearum, which is capable of forming dipeptide bond as confirmed by the release of phosphoric acid (Kino K. et al., Biochem. Biophys. Res. Comm., 2008, 371:536-540; European Patent Application No. 1870454). The structural analysis using NMR technique confirmed formation of dipeptides having L-Ser, L-Met, L-Gln, L-Phe, L-His, L-Ala, and L-Cys at the N-terminus. Despite the inorganic phosphate release has been confirmed in the mixture containing RSp1486a and L-Asp with L-Phe, L-His, L-Met, L-Cys or L-Ala; or RSp1486a and L-Glu with L-Phe, L-His, L-Met, L-Cys, L-Ser, or L-Ala, the structural analysis of reaction products has not been performed. No additional phosphoric acid release above background level has been observed in reaction mixture containing RSp1486a and L-Asp or L-Glu. A newly discovered L-amino acid ligase RizB from B. subtilis NBRC3134 has been found to synthesize various heteropeptides and homo-oligomers of branched-chain amino acids consisting of 2 to 5 amino acid residues (Kino K., Yakugaku Zasshi, 2010, 130(11):1463-1469). For example, formation of dimer, trimer, and tetramer of L-Val has been proven by LC-ESI-MS analysis in the mixture containing RizB, L-Val, and L-Glu or L-Asp. No heteropeptides have been revealed.